Abstract
The limited efficacy of cancer immunotherapy occurs due to the lack of
spatiotemporal orchestration of adaptive immune response stimulation
and immunosuppressive tumor microenvironment modulation. Herein, we
report a nanoplatform fabricated using a pH-sensitive triblock
copolymer synthesized by reversible addition-fragmentation chain
transfer polymerization enabling in situ tumor vaccination and
tumor-associated macrophages (TAMs) polarization. The nanocarrier
itself can induce melanoma immunogenic cell death (ICD) via tertiary
amines and thioethers concentrating on mitochondria to regulate
metabolism in triggering endoplasmic reticulum stress and upregulating
gasdermin D for pyroptosis as well as some features of ferroptosis and
apoptosis. After the addition of ligand cyclic
arginine-glycine-aspartic acid (cRGD) and mannose, the mixed
nanocarrier with immune adjuvant resiquimod encapsulation can target
B16F10 cells for in situ tumor vaccination and TAMs for M1 phenotype
polarization. In vivo studies indicate that the mixed targeting
nanoplatform elicits tumor ICD, dendritic cell maturation, TAM
polarization, and cytotoxic T lymphocyte infiltration and inhibits
melanoma volume growth. In combination with immune checkpoint blockade,
the survival time of mice is markedly prolonged. This study provides a
strategy for utilizing immunoactive materials in the innate and
adaptive immune responses to augment cancer therapy.
Subject terms: Molecular medicine, Drug delivery, Cancer immunotherapy
__________________________________________________________________
Cancer immunotherapy leans on the effective activation on immune
response within tumors and surrounding immunosuppressive
microenvironment. Here, the authors report a triblock copolymeric
nanocarrier encapsulating Resiquimod for B16F10 cell-targeting, which
enables in situ tumor vaccination and tumor-associated macrophages
polarization.
Introduction
Cancer immunotherapy is one of the most promising strategies for tumor
treatment and has attracted extensive attention. Induction of tumor
immunogenic cell death (ICD) is a widely used methodology in which
tumor cells, after encountering external stimuli, can change from a
non-immunogenic state to an immunogenic state with host immune response
provocation^[42]1–[43]3. The typical immune features of ICD are
damage-associated molecular patterns (DAMPs) (e.g., calreticulin [CRT],
high mobility group box 1 [HMGB1], and adenosine triphosphate [ATP])
secretion^[44]4,[45]5 and tumor-associated antigen release that can
facilitate dendritic cell (DC) maturation, migration, and antigen
presentation to T cells for host immunity activation^[46]6,[47]7.
Currently, various strategies, including photodynamic therapy,
chemotherapy, and pyroptosis, have been developed for ICD-mediated
tumor immunotherapy^[48]1,[49]8–[50]15. However, these methodologies
generally require additional ICD inducers that may complicate the
nanoplatform. Therefore, the development of nanocarriers that can
directly induce ICD is imperative to simultaneously simplify
nanoformulations to ensure therapeutic efficacy due to their own
immunity.
Tumor vaccines have emerged as a promising strategy for long-term
cancer immunotherapy, and they primarily attack tumors via eliciting an
antigen-specific immune response^[51]16. Although there are definite
antigens in tumor vaccines, the heterogeneity and high cost of mutant
antigen identification have restricted their further
development^[52]17,[53]18. To cope with the above issues, in situ tumor
vaccination can be greatly enhanced via combining immune adjuvants with
tumor-associated antigens (TAAs) that can be directly released by dying
tumor cells in vivo after different treatments (e.g., photodynamic
therapy, photothermal therapy, and chemotherapy)^[54]14,[55]19–[56]26.
The most obvious advantage of in situ tumor vaccination is that extra
antigen addition is unnecessary, and it depends on the antigen pool
from individual tumors after external stimulation, thereby saving
valuable time and cost.
Although eliciting adaptive immunity is an extensively used strategy
for cancer immunotherapy, immunosuppressive factors in the tumor
microenvironment (TME) (e.g., tumor-associated macrophages [TAMs]) can
greatly impede therapeutic efficacy^[57]27–[58]31. TAMs primarily exist
as an immunosuppressive M2 phenotype and account for 50% of all immune
cells in tumor tissue^[59]32, and they help tumors to escape
immunosurveillance via generating anti-inflammatory
cytokines^[60]33–[61]35. In contrast, M1 phenotype TAMs act as
pro-inflammatory cells and can eliminate tumors with antigen
presentation to T lymphocytes, thereby polarizing M2-TAMs into the M1
phenotype necessary^[62]36–[63]38. Resiquimod (R848) can induce TAM
polarization^[64]39; however, its application is limited by severe side
effects after systemic injection^[65]32. Nanomedicine that achieves
high tumor accumulation via the enhanced permeability and retention
effect can improve drug bioavailability with effective drug delivery
and negligible side effects. Especially, the different targeting
ligands decoration on nanosystems can separately target different cells
such as tumor, TAMs, etc. for spatiotemporal orchestration.
Additionally, the high expression of cluster of differentiation 47
(CD47) on the tumor cell surface contributes to tumor resistance to
macrophage phagocytosis via the CD47/signal regulatory protein alpha
(SIRPα) axis^[66]40. Thus, both TAM polarization and blockade of the
immune checkpoint molecule CD47 are necessary for immunosuppressive TME
modulation.
In this work, we report an immunofunctional nanoplatform in which the
nanocarrier itself directly induces tumor ICD via metabolic regulation
of elevated oxidative phosphorylation and via gasdermin D (GSDMD)
upregulation for pyroptosis. After conjugation of the targeting ligand
cyclic arginine-glycine-aspartic acid (cRGD) or mannose (Man) to the
same polymeric skeleton, the mixed nanocarrier separately targets tumor
cells and TAMs in vivo. After encapsulation with the immune adjuvant
R848, cRGD-decorated nanoparticles selectively target melanoma B16F10
cells for carrier-mediated ICD to form an in situ cancer vaccine, and
Man-modified nanoparticles target TAMs, ultimately resulting in
polarization for TME modulation. In vivo results indicate that the
mixed-targeting nanoformulation elicits DC maturation, M1 like TAM
polarization, CD8^+/CD4^+ T cell infiltration, and melanoma tumor
volume growth inhibition (Fig. [67]1). After combination treatment with
anti-PD-1 and anti-CD47, the formulation notably prolongs the median
survival of mice and results in tumor cell death. This project
highlights the mechanism of polymer-mediated ICD and provides insights
into the exploitation of immunoactive nanomaterials to spatiotemporally
orchestrate adaptive and innate immune responses for effective cancer
immunotherapy.
Fig. 1. Schematic illustration of cRGD- mix Man-pRNC[Thioether+DEA]@R848
mediated in situ tumor vaccination and TAMs polarization with nanocarrier
itself as the tumor ICD inducer.
[68]Fig. 1
[69]Open in a new tab
cRGD-pRNC[Thioether+DEA] selectively targeting B16F10 tumor induces
metabolism regulation with oxidative phosphorylation elevation and
activates GSDMD with pyroptosis for tumor ICD. After combination with
R848, cRGD-pRNC[Thioether+DEA]@R848 forms in situ tumor vaccination.
Man-pRNC[Thioether+DEA]@R848 specifically targets TAMs leading to
polarization into M1 phenotype with iNOS, CD80 expression and IL-12
secretion increment. After combination with anti-PD-1 and anti-CD47,
the mixed nanoformulation elicits antitumor immune response with Treg
decrement and CD8^+/CD4^+ T cell proliferation increment.
Results
Synthesis of functional polymers
The successful synthesis of polymers is a prerequisite for constructing
nanoplatforms. Before the synthesis of the skeleton polymer,
polyethylene glycol-poly methyl methacrylate (PEG-PMMA), the
macro-reversible addition-fragmentation chain transfer (RAFT)
polymerization agent PEG-CPPA was synthesized via an amidation reaction
between PEG-NH[2] and NHS-CPPA (Supplementary Fig. [70]1). The
conversion ratio of NHS-CPPA that was the abbreviation of a small
molecule RAFT agent known as
4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester
was 98% based on peaks of methylene protons in PEG (δ, 3.63 ppm) and
methine protons in CPPA (δ, 7.38, 7.57, and 7.90 ppm) of ^1H NMR
spectra (Supplementary Fig. [71]3). Di-block copolymer PEG-PMMA (named
as P[MMA]) was obtained through RAFT polymerization of PEG-CPPA and
monomer MMA, and the molecular weight was 5.0-10.0 kg/mol according to
the peaks of PEG protons (δ, 3.63 ppm) and methyl protons in PMMA (δ,
3.65 ppm) of ^1H NMR spectra. GPC indicated that the relative molecular
weight was 15.3 kg/mol with a polydispersity index (PDI) of 1.3
(Fig. [72]2a, b, Supplementary Fig. [73]4 and Supplementary
Table [74]1). Tri-block copolymer PEG-PMMA-PDEA (termed as P[DEA]) was
synthesized by PEG-PMMA and monomer diethylaminoethyl methacrylate
(DEA) via RAFT polymerization (Supplementary Fig. [75]1), and the
molecular weight was 5.0-10.0-3.8 kg/mol based on methylene protons in
PEG (δ, 3.63 ppm) and methylene protons in PDEA (δ, 0.86 and 1.26 ppm)
of ^1H NMR spectra. GPC indicated that the relative molecular weight
was 19.2 kg/mol (Fig. [76]2a, b, Supplementary Fig. [77]5 and
Supplementary Table [78]1). The small molecule N-propargyl
methacrylamide (PPMA) was obtained from methacryloyl chloride and
propargylamine (Supplementary Fig. [79]2), and it was pure according to
^1H NMR result based on protons at δ 1.97 ppm, δ 2.25 ppm, δ 4.10 ppm,
δ 5.37 ppm, and δ 5.74;ppm (Supplementary Fig. [80]6). To obtain
PEG-PMMA-P (PPMA-ME) (termed P[Thioether]), tri-block copolymer
PEG-PMMA-PPPMA (termed P[yne]) was first synthesized using PEG-PMMA and
PPMA via RAFT polymerization, and the molecular weight was
5.0-10.0-6.3 kg/mol based on peaks of methylene protons in PEG (δ,
3.63 ppm), methyl protons in PMMA (δ, 3.65 ppm), and methyl protons in
PPPMA (δ, 1.81 ppm) of ^1H NMR spectra. GPC indicated that the relative
molecular weight was 18.9 kg/mol (Fig. [81]2a, b, Supplementary
Fig. [82]7 and Supplementary Table [83]1). After a click reaction of
alkynyl in PPPMA with mercaptoethanol (ME) (Supplementary Fig. [84]2),
PEG-PMMA-P(PPMA-ME) (P[Thioether]) was obtained, and the graft ratio of
thiol was 94% based on peaks of PEG protons at δ 3.63 ppm, methyl
protons in PMMA (δ, 3.65 ppm), and methylene protons at δ 2.89 ppm and
δ 2.97 ppm. GPC indicated that the relative molecular weight was
30.7 kg/mol (Supplementary Fig. [85]8 and Supplementary Table [86]1).
PEG-PMMA-P(PPMA-MPA) was synthesized from PEG-PMMA-PPPMA and
mercaptopropionic acid (MPA) via a click reaction after further
esterification with diethylaminoethanol to obtain
PEG-PMMA-P(PPMA-MPA-DEA) (termed P[Thioether+DEA]) (Supplementary
Fig. [87]2). ^1H NMR results revealed that the molecular weight of
P[Thioether+DEA] was 5.0-7.3-18.4 kg/mol based on peaks of PEG protons
at δ 3.63 ppm, methyl protons in PMMA (δ, 3.65 ppm), methylene protons
(δ, 0.86 ppm), and methyl groups (δ, 1.25 ppm) in P(PPMA-MPA-DEA). GPC
indicated that the relative molecular weight was 36.1 kg/mol
(Supplementary Figs. [88]9–[89]11 and Supplementary Table [90]1).
Fig. 2. Preparation and characterization of the ICD inducer
pRNC[Thioether+DEA].
[91]Fig. 2
[92]Open in a new tab
a Structures of a series of functional polymers with the same skeleton.
b ^1H NMR spectra of different polymers. c Size and transmission
electron microscopy images of NC[MMA], NC[yne], pRNC[DEA],
NC[Thioether], pRNC[Thioether+DEA]. Scale bar = 100 nm. Three times
each experiment was repeated independently with similar results. d CRT
exposure after different treatments via flow cytometry
characterization. e CRT exposure and HMGB1 release of B16F10 cells
after different treatments characterized by CLSM. Cells were stained
with Alexa 488-anti-CRT (green) and Alexa 488-anti-HMGB1 (green). Cell
nuclei were stained with DAPI (blue). Scale bar = 20 μm. f
Semi-quantitative analysis of CRT exposure after different treatments
(n = 3 independent experiments). g pRNC[Thiorther+DEA] with different
concentrations induced CRT exposure in B16F10 cells (n = 3 independent
experiments). h CRT exposure of B16F10 cells was induced by
pRNC[Thioether+DEA] after different incubation time treatment (n = 3
independent experiments). i The kinetics and dose-response of cell
death induction by pRNC[Thioether+DEA] (n = 3 independent experiments).
Data are presented as mean ± SD. Statistical significance was
calculated through one-way ANOVA for multiple comparisons using a Tukey
post-hoc test.
Selection of polymers that specifically induce B16F10 ICD
Nanocarrier self-assemblies from PEG-PMMA, PEG-PMMA-PPPMA,
PEG-PMMA-PDEA, PEG-PMMA-P(PPMA-ME), and PEG-PMMA-P(PPMA-MPA-DEA)
(Fig. [93]2a, b) were prepared using the solvent exchange method and
separately named NC[MMA], NC[yne], pRNC[DEA], NC[Thioether], and
pRNC[Thioether+DEA]. Dynamic light scattering (DLS) results indicated
that the nanocarriers possessed a hydrodynamic diameter varying from
120 to 150 nm and a PDI varying from 0.18 to 0.25 (Fig. [94]2c,
Supplementary Table [95]2). The morphology of pRNC[Thioether+DEA] was
characterized by transmission electron microscopy (TEM). As presented
in Fig. [96]2c, it is a uniformly spherical polymer with a hollow
structure. In the acetate buffer solution (pH 5.0, 10 mM), the size and
PDI of pRNC[Thioether+DEA] and pRNC[DEA] increased over time, whereas
no obvious changes were observed in the PBS buffer with a pH value of
7.4, thus highlighting their pH responsiveness and physiological
stability (Supplementary Fig. [97]12). Additionally, both
pRNC[Thioether+DEA] and pRNC[DEA] maintained good stability under
weakly acidic condition (pH 6.5) that mimicked the TME (Supplementary
Fig. [98]13).
Their ability to induce ICD was investigated by CRT exposure and HMGB1
and ATP release via flow cytometry (FCM), confocal laser scanning
microscopy (CLSM), and ATP assays. The FCM results presented in
Fig. [99]2d, f indicated that NC[MMA] and NC[yne] did not induce CRT
exposure, and both displayed similar CRT-positive ratios to that of
phosphate-buffered saline (PBS). Slightly elevated ratios were detected
in the pRNC[DEA] (14.70 ± 0.17%) and NC[Thioether] (14.23 ± 0.90%)
groups, and the highest ratio (19.63 ± 0.55%) was observed in the
pRNC[Thioether+DEA] group, thus indicating that both tertiary amine and
thioether were able to induce ICD. Compared to pRNC[DEA] and
NC[Thioether], pRNC[Thioether+DEA] induced the highest CRT exposure
that was 1.33- to 1.37-fold higher than that of the other two groups.
This was likely due to the dual ICD effects of both DEA and thioether
groups (Fig. [100]2d, f, Supplementary Fig. [101]14). CLSM results
displayed a similar tendency to those of FCM, where cells treated with
pRNC[Thioether+DEA] exhibited the most obvious CRT exposure (green)
compared to that of the pRNC[DEA] and NC[Thioether] groups
(Fig. [102]2e). Moreover, HMGB1 (green) in cells treated with NC[MMA]
and NC[yne] overlapped well with the nuclei, whereas HMGB1 was
primarily distributed outside of the nuclei in the pRNC[DEA] and
NC[Thioether] groups, thus indicating that both nanocarriers could
induce HMGB1 release from the nuclei. After pRNC[Thioether+DEA]
treatment, the green color of HMGB1 outside the nucleus decreased, thus
indicating the robust ability of this nanocarrier to induce ICD
(Fig. [103]2e). Additionally, after different treatments,
pRNC[Thioether+DEA] induced the highest HMGB1 release into the cell
supernatant (Supplementary Fig. [104]15a). According to the ATP
detection results, cells treated with pRNC[Thioether+DEA] possessed the
highest ATP levels in the supernatant (Supplementary Fig. [105]15b).
Therefore, based on the above results, pRNC[Thioether+DEA] was the best
ICD inducer and was selected as the optimal nanoparticle for further
studies. We then investigated the effects of factors such as
concentration and incubation time on pRNC[Thioether+DEA]-mediated ICD.
We observed that CRT exposure increased with pRNC[Thioether+DEA]
concentration increment at the same time point. The highest amount of
CRT exposure was detected at 100 μg/mL, and it was 2.37- to 3.61-fold
higher than that of the others (Fig. [106]2g). Thus,
pRNC[Thioether+DEA]at 100 μg/mL was chosen as the ICD inducer for the
following experiments. As presented in Fig. [107]2h, the CRT exposure
increased with prolonged incubation time, and the maximum exposure was
observed at 48 h. This exposure was 1.46- to 2.18-fold higher than that
of the other time points. The results indicated that
pRNC[Thioether+DEA]-mediated ICD was dependent upon both concentration
and incubation time. As presented in Fig. [108]2i,
pRNC[Thioether+DEA]-mediated B16F10 cell death was
concentration-dependent with an IC[50] value of 93.04 µg/mL. In
addition to B16F10 cells, pRNC[Thioether+DEA] induced ICD in multiple
cancer cells such as colorectal carcinoma MC38, Lewis lung cancer (LLC)
cells, and pancreatic carcinoma Pan02 with a notable CRT fluorescence
shift when slight CRT exposure was observed in breast cancer 4T1 and
glioma U87-MG cells (Supplementary Fig. [109]16). These results
revealed that pRNC[Thioether+DEA] can be widely applied to a variety of
tumors for immunotherapy via inducing ICD.
Investigation of the pRNC[Thioether+DEA]-mediated ICD mechanism
Inspired by the observation that pRNC[Thioether+DEA] could induce
B16F10 ICD, the underlying mechanism was investigated via the
intracellular co-localization of nanocarriers with organelles and by
single-cell RNA sequencing (scRNA-seq) analysis. To conveniently
observe the intracellular distribution of the nanocarrier,
pRNC[Thioether+DEA] was labelled with Cy5 (Cy5-pRNC[Thioether+DEA]),
observed, and then captured using CLSM. Cy5-pRNC[Thioether+DEA] first
reached the endo/lysosome at 2 h and then escaped at 4 h (Supplementary
Fig. [110]17a, b). According to Fig. [111]3a, it was primarily
concentrated in the mitochondria at 4 h and exhibited obvious
co-localization, while no obvious Cy5 fluorescence was observed in the
endoplasmic reticulum (Fig. [112]3b) or Golgi apparatus (Supplementary
Fig. [113]17c). These results demonstrate that pRNC[Thioether+DEA] was
primarily concentrated in the mitochondria of tumor ICD.
Fig. 3. Mechanism investigation of pRNC[Thioether+DEA] mediated B16F10 ICD.
[114]Fig. 3
[115]Open in a new tab
a, b Representative images of pRNC[Thioether+DEA] mediated
co-localization with mitochondria and ER. MitoTracker green (a) and ER
Tracker green (b) were stained with mitochondria and ER, respectively.
Red color represented Cy5-pRNC[Thioether+DEA], and blue color
represented DAPI which were stained by Hoechst 33342. PCC = Pearson’s
correlation coefficient. Scale bar = 10 μm. c Volcano map of expressed
differentially genes in pRNC[Thioether+DEA] treated cells. d Heatmap
depicting relative transcript levels of expressed differentially genes
in pRNC[Thioether+DEA] or PBS treated cells (n = 3 independent
experiments). e KEGG pathway enrichment analysis of expressed
differentially genes for cells after pRNC[Thioether+DEA] treatment. f,
g Enrichment of organismal systems pathways (f) and metabolism pathways
(g) in pRNC[Thioether+DEA] or PBS treated cells (n = 3 independent
experiments). h Intracellular ROS level after different treatments via
flow cytometer characterization (n = 3 independent experiments). i
Intracellular mtROS level after treatments via CLSM characterization.
Scale bar = 20 μm. j Western blot of p-PERK, p-elf2α, ATF4, cleaved
caspase-1, N-GSDMD, MLKL expression after treatments. β-actin was used
as internal control. Experiments in (a), (b), (i), and (j) were
repeated three times independently with similar results.
It has been reported that both metabolic regulation and redox
homeostasis disruption in mitochondria can mediate tumor ICD via
eliciting oxidative phosphorylation^[116]41–[117]44. To the best of our
knowledge, this is the first study to show that polymeric nanocarriers
with specific structures can induce tumor ICD via mitochondria
metabolism regulation with ER stress and GSDMD activation with
pyroptosis. As indicated in the scRNA-seq results, the mitochondrial
cytochrome c oxidase subunit 3 gene (mt-co3) and GSDMD displayed
significant differences according to the volcano image for B16F10 cells
treated with pRNC[Thioether+DEA] (Fig. [118]3c). Obvious mt-co3
downregulation and perp (a mediator of p53 dependent apoptosis)
upregulation were detected, thus demonstrating that pRNC[Thioether+DEA]
could also induce tumor apoptosis (Fig. [119]3c, d). As presented in
Fig. [120]3c, multiple genes related to chemokines (e.g., chemokine
[C-X-C motif] ligand 11 [CXCL11]) and GSDMD were upregulated after
pRNC[Thioether+DEA] treatment, and this activated multiple downstream
immune-related signaling pathways as well as pyroptosis. Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
demonstrated that the dominant pathways enriched by pRNC[Thioether+DEA]
were NOD-like receptor, p53, and the TNF signaling pathway as well as
oxidative phosphorylation that would mediate a series of downstream
immune and inflammatory cascades, apoptosis, and metabolism regulation
(Fig. [121]3e). According to the KEGG regulatory network diagram,
metabolic pathways, particularly those related to oxidative
phosphorylation, were significantly different (Fig. [122]3g). Multiple
immune-related regulatory pathways, including NOD-like receptor,
chemokine, and IL-17 signaling pathways, were activated after
pRNC[Thioether+DEA] treatment (Fig. [123]3f).
As presented in Fig. [124]3h, the intracellular ROS level detected
using DCFH-DA as the fluorescent probe was notably elevated in cells
treated with pRNC[Thioether+DEA]. Therefore, the destruction of redox
homeostasis likely elicited tumor ICD. Compared to NC[MMA],
pRNC[Thioether+DEA] cells exhibited a 1.5-fold increase in
intracellular ROS fluorescence intensity (Fig. [125]3h, Supplementary
Fig. [126]18). To further determine if the elevated intracellular ROS
was produced by mitochondria, we used CLSM to observe the production of
intracellular mitochondrial ROS (mtROS) using BBcellProbe® OM08 as a
detection probe after 24 h treatment. Notable red fluorescence was
observed in the pRNC[Thioether+DEA]-treated group compared to that in
the PBS and NC[MMA] groups (Fig. [127]3i, Supplementary Fig. [128]19),
thus indicating that pRNC[Thioether+DEA] induced mtROS generation.
To further investigate how pRNC[Thioether+DEA] induces ICD in B16F10
cells, we used western blotting to examine the ER stress-related
pathways. The phosphorylation of PERK and eukaryotic translation
initiation factor 2α (elf2α) was enhanced after pRNC[Thioether+DEA]
treatment at 48 h compared to that in response to PBS and NC[MMA]. The
levels of downstream activating transcription factor 4 (ATF4) and
recombinant DNA damage-inducing transcript 3 (CHOP) were also
increased. These data indicate that ER stress was triggered by
pRNC[Thioether+DEA] (Fig. [129]3j, Supplementary Fig. [130]20). To
explore pRNC[Thioether+DEA] mediated cell death, B16F10 cells after
different treatments were stained with calcein-AM and propidium iodide
(PI) to identify live/dead cells. The noticeable increase of red spots
(dead cells) was observed in pRNC[Thioether+DEA] group while a large
numbers of surviving cells (green color) were detected in PBS and
NC[MMA] groups, indicating the ability of pRNC[Thioether+DEA] to induce
cell death (Supplementary Fig. [131]21). The cell death types were then
investigated in consideration that many types such as pyroptosis,
apoptosis and ferroptosis etc. can induce tumor ICD. The typical
pyroptotic morphologies were observed in cells after
pRNC[Thioether+DEA]treatment indicating its ability to induce
pyroptosis (Supplementary Fig. [132]22). Additionally, the expression
of N-GSDMD and cleaved caspase-1 was upregulated after
pRNC[Thioether+DEA] treatment, and lactate dehydrogenase (LDH) activity
was 2.4-fold higher than that in PBS, indicating that
pRNC[Thioether+DEA] induces pyroptosis through the GSDMD pathway
(Fig. [133]3j, Supplementary Fig. [134]23). According to Supplementary
Fig. [135]24, pRNC[Thioether+DEA] also induced apoptosis when the
elevated early (10.50 ± 0.46%) and late (55.27 ± 4.38%) apoptosis
ratios were detected compared to those of the PBS (early: 4.70 ± 0.14%;
late: 14.20 ± 0.98%) and NC[MMA] (early: 5.27 ± 0.56%; late:
19.23 ± 0.93%) groups. Additionally, we observed that oxidized lipid
peroxide (LPO) levels were elevated after pRNC[Thioether+DEA]
treatment, thus indicating its ability to induce cancer ferroptosis.
The ratio of oxidized to reduced LPO was increased to 40.01 ± 0.12%
compared to that of PBS (30.97 ± 0.32%) and NC[MMA] (29.60 ± 0.24%)
(Supplementary Fig. [136]25, [137]26). CLSM results in Supplementary
Fig. [138]26 further confirmed the LPO generation when the notable
increment of oxidized C11-BODIPY (green color) was observed for cells
with pRNC[Thioether+DEA] treatment. Moreover, negligible changes in the
expression of mixed lineage kinase domain-like protein (MLKL) were
observed after treatment with PBS, NC[MMA], or pRNC[Thioether+DEA]. The
existence of necrostatin 2 racemate (Nec-1s) did not affect MLKL
expression, further confirming that pRNC[Thioether+DEA] did not induce
cell necroptosis (Fig. [139]3j, Supplementary Fig. [140]27). To further
distinguish cell death types when detecting the cell death populations,
inhibitors such as Z-VAD-FMK for apoptosis, Ferrostatin-1 (Fer-1) for
ferroptosis, and Nec-1s for necroptosis were added to B16F10 cells
treated with pRNC[Thioether+DEA]. As shown in Supplementary
Fig. [141]28, the ratio of dead/dying cells induced by
pRNC[Thioether+DEA] was highly suppressed after an apoptosis inhibitor
Z-VAD-FMK treatment. After treatment with the ferroptosis inhibitor
Ferrostatin-1 (Fer-1), the cell death ratio slightly reduced, while
negligible changes were observed in cells treated with the necroptosis
inhibitor necrostatin 2 racemate (Nec-1s). The data indicated that
pRNC[Thioether+DEA] induced mixed cell death modalities with some
features of pyroptosis, ferroptosis, and apoptosis.
Preparation and characterization of cRGD-pRNC[Thioether+DEA] and
Man-pRNC[Thioether+DEA]
To target B16F10 cells and TAMs separately, cRGD- and Man-targeting
ligands were decorated onto the nanocarrier surface to obtain
cRGD-pRNC[Thioether+DEA] and Man-pRNC[Thioether+DEA], respectively[.]
c(RGDfK) is a cyclic peptide that can target tumor cells (e.g., B16F10
cells, LLC cells) with high expression of α[v]β[3], and Man is a cyclic
monosaccharide that targets TAMs with high mannose receptor
expression^[142]45–[143]47. The synthesis of cRGD (Man)-PEG-PMMA-PPPMA
was similar to that of PEG-PMMA-PPPMA (Supplementary Fig. [144]29). The
ratios of c(RGDfK) and Man were 85% and 87%, respectively, based on
peaks of methylene protons in PEG (δ, 3.63 ppm), methylene protons (δ,
1.84 ppm) in cRGD (Supplementary Fig. [145]30), and methine proton (δ,
2.28 ppm) in Man according to ^1H NMR results (Supplementary
Fig. [146]31).
cRGD-pRNC[Thioether+DEA] and Man-pRNC[Thioether+DEA] were also
fabricated via the solvent-exchange method. DLS results indicated that
their diameter sizes were 168.0 ± 4.85 nm and 143.5 ± 1.08 nm,
respectively, and PDI was 0.16 ± 0.016 and 0.23 ± 0.021 (Fig. [147]4a,
b, Supplementary Table [148]3). After mixing, cRGD- mix
Man-pRNC[Thioether+DEA] was obtained, and no obvious changes were
detected (Supplementary Fig. [149]32 and Supplementary Table [150]3).
According to Fig. [151]2f, the concentration of pRNC[Thioether+DEA] as
the ICD inducer was chosen as 100 μg/mL due to the obvious CRT
exposure, and this concentration was also suitable for
cRGD-pRNC[Thioether+DEA]. According to Fig. [152]4c, the cell viability
of RAW264.7 cells was 88 ± 2.8% when the Man-pRNC[Thioether+DEA]
concentration was 50 μg/mL, while the value decreased to 73 ± 2.6% when
the concentration was increased to 100 μg/mL. Thus, to reduce
macrophage death the concentration of Man-pRNC[Thioether+DEA] was
selected as 50 μg/mL. cRGD (Man)-pRNC[Thioether+DEA] with R848
encapsulation (cRGD (Man)-pRNC[Thioether+DEA]@R848) was also separately
prepared via the solvent-exchange method when the drug loading content
(DLC) was as high as 12.70% and 13.29% with drug loading efficiency
(DLE) of 58.2% and 61.3%, respectively, according to a
fluorospectrophotometer (Supplementary Fig. [153]33). The DLC and DLE
values of the cRGD- mix Man-pRNC[Thioether+DEA] were 12.55% and 57.4%,
respectively (Supplementary Table [154]3). In vitro drug release
behavior indicated that the accumulative release of R848 from
cRGD-pRNC[Thioether+DEA]@R848, Man-pRNC[Thioether+DEA]@R848, and cRGD-
mix Man-pRNC[Thioether+DEA]@R848 was as high as 61%, 60%, and 67.6%
within 24 h in acetate buffer solution (pH 5.0, 10 mM, 150 mM NaCl),
while the release was only 23%, 24%, and 20.2% in PBS (pH 7.4, 10 mM,
150 mM NaCl) (Fig. [155]4d, e, Supplementary Fig. [156]34). The above
results indicated that cRGD (Man)-pRNC[Thioether+DEA]@R848 as well as
the mixed nanoformulations were pH-sensitive.
Fig. 4. Preparation and characterization of targeted nanoformulations.
[157]Fig. 4
[158]Open in a new tab
a, b Diameters and representative transmission electron microscopy
images of cRGD-pRNC[Thioether+DEA] and Man-pRNC[Thioether+DEA]. Scale
bar, 100 nm. Three times was repeated independently with similar
results. c Dosage dependent cytotoxicity of Man-pRNC[Thioether+DEA] in
RAW264.7 cells by MTT assays characterization. Data are shown as
mean ± SD (n = 3 independent experiments). d, e In vitro R848 release
from cRGD-pRNC[Thioether+DEA] or Man-pRNC[Thioether+DEA] within 24 h at
different pH values (pH 7.4 or pH 5.0). Data are shown as Geometric
mean ± 95% CI. (n = 3 independent experiments). f Representative flow
cytometric plots showing the cellular uptake of pRNC[Thioether+DEA],
cRGD-pRNC[Thioether+DEA] and free FITC in B16F10 cells. g
Representative flow cytometric plots showing the cellular uptake of
pRNC[Thioether+DEA], Man-pRNC[Thioether+DEA] and free FITC in RAW264.7
cells. h, i Representative flow cytometric images and quantification of
mature DCs. Data are shown as mean ± SD (n = 3 independent
experiments). j, k Representative flow cytometric images and
quantification of polarization of macrophages. Data are shown as
mean ± SD (n = 3 independent experiments). l Expression of iNOS in
cells after different treatments (n = 3 independent experiments). The
word “ns” represented non-significance, and ^*P < 0.05; ^**P < 0.01;
^***P < 0.001. Data are presented as mean ± SD. Statistical
significance was calculated through one-way ANOVA for multiple
comparisons using a Tukey post-hoc test.
In vitro targetability of cRGD-pRNC[Thioether+DEA] and
Man-pRNC[Thioether+DEA]
R848 was replaced with FITC to better monitor intracellular
internalization that was characterized by FCM. As presented in
Fig. [159]4f, a more obvious fluorescence shift and cytotoxicity were
observed in B16F10 cells in response to cRGD-pRNC[Thioether+DEA]@FITC
treatment than that in response to pRNC[Thioether+DEA]@FITC, thus
indicating the good targetability of cRGD (Supplementary Figs. [160]35,
[161]37). After Man-pRNC[Thioether+DEA]@FITC treatment, a stronger
fluorescence shift and cytotoxicity were observed in RAW264.7 cells
compared to the pRNC[Thioether+DEA]@FITC group, thus indicating
noticeable targetability of Man (Fig. [162]4g, Supplementary
Fig. [163]36). The CLSM results in Supplementary Fig. [164]38 indicated
that cRGD and Man decoration enhanced the targetability of B16F10 and
RAW264.7, respectively.
cRGD-pRNC[Thioether+DEA]@R848 mediated DCs maturation
To test if the nanocarrier could activate DCs in vitro, we first
examined its ability to induce ICD in B16F10 cells as characterized by
FCM. An elevated CRT^+ ratio was observed in cells treated with
cRGD-pRNC[Thioether+DEA] or cRGD-pRNC[Thioether+DEA]@R848 compared to
that of NC[MMA] and PBS. Negligible differences were observed among the
cRGD-pRNC[Thioether+DEA], cRGD-pRNC[Thioether+DEA]@R848, and cRGD- mix
Man-pRNC[Thioether+DEA]@R848 treated groups, thus indicating that
cRGD-pRNC[Thioether+DEA] induced ICD while R848 did not and that the
mixing of cRGD- and Man-nanoformulations did not affect the ICD
inducibility of cRGD-pRNC[Thioether+DEA] (Supplementary Fig. [165]39).
When B16F10 cell supernatant was added into DCs, it was observed that
both cRGD-pRNC[Thioether+DEA] (CD11c^+CD80^+: 20.30 ± 2.25%;
CD11c^+CD86^+: 23.60 ± 2.95%) and cRGD-pRNC[Thioether+DEA]@R848
(CD11c^+CD80^+: 19.80 ± 0.20%; CD11c^+CD86^+: 23.30 ± 0.36%) treated
cancer cells displayed a notable DC maturation compared to that of
NC[MMA] (CD11c^+CD80^+: 9.53 ± 0.27%; CD11c^+CD86^+: 12.80 ± 0.78%) and
PBS (CD11c^+CD80^+: 9.56 ± 1.24%; CD11c^+CD86^+: 13.00 ± 1.05%) that
was similar to that of the positive control LPS (CD11c^+CD80^+:
24.20 ± 0.87%; CD11c^+CD86^+: 28.07 ± 0.70%) (Fig. [166]4h, i,
Supplementary Fig. [167]40). This suggests that tumor ICD mediated by
cRGD-pRNC[Thioether+DEA] with DAMP secretion facilitates DC maturation.
Additionally, the cRGD- mix Man-pRNC[Thioether+DEA]@R848-treated group
exhibited similar ratios of CD11c^+CD80^+ (20.20 ± 0.87%) and
CD11c^+CD86^+ (23.60 ± 1.13%) to that of cRGD-pRNC[Thioether+DEA]@R848,
thus demonstrating that the mixing of cRGD and Man nanoformulations did
not affect the cRGD-pRNC[Thioether+DEA]-mediated ICD cascade with DC
maturation. As presented in Supplementary Fig. [168]41, both the
cRGD-pRNC[Thioether+DEA] and cRGD-pRNC[Thioether+DEA]@R848 groups
exhibited lower ratios of DCs to dead B16F10 cells in the supernatants
compared to that of PBS and NC[MMA], indicating that these two groups
induced more dead cells for DAMPs generation with DC maturation.
Man-pRNC[Thioether+DEA]@R848-mediated macrophage polarization
To determine if Man-pRNC[Thioether+DEA]@R848 could induce macrophage
polarization, the expression of CD206 and CD80 on the surface of
RAW264.7 cells and intracellular inducible nitric oxide synthase (iNOS)
levels were detected. As presented in Fig. [169]4j, k, CD80 expression
on the cell surface after Man-pRNC[Thioether+DEA]@R848 treatment was
14.33 ± 0.83%, and this was 2.4-fold higher than that of
Man-pRNC[Thioether+DEA] (6.63 ± 0.08%) and similar to that of free R848
(17.67 ± 5.95%). CD206 expression in cells with
Man-pRNC[Thioether+DEA]@R848 treatment decreased to 14.53 ± 0.47%, and
this was 1.75-fold lower than that of Man-pRNC[Thioether+DEA]
(25.47 ± 0.21%). According to the results of quantitative polymerase
chain reaction (qPCR), R848 could promote the expression of iNOS while
Man-pRNC[Thioether+DEA] alone not. Man-pRNC[Thioether+DEA]@R848 induced
notable iNOS expression that was 4.78-fold higher than that of
Man-pRNC[Thioether+DEA] (1.01 ± 0.3) (Fig. [170]4l). According to the
above results, pRNC[Thioether+DEA]@R848 can polarize M2 RAW264.7 cells
into an M1 phenotype similar to that caused by free R848.
In vivo targetability of cRGD-pRNC[Thioether+DEA] and Man-pRNC[Thioether+DEA]
To avoid fluorescence interference from melanoma, B16F10 cells were
replaced with LLC cells to construct a tumor model in C57BL/6 mice for
in vivo targeting of cRGD-pRNC[Thioether+DEA] and
Man-pRNC[Thioether+DEA] via a near-infrared (NIR) in vivo imaging
system (IVIS), FCM, and immunofluorescence staining analysis.
cRGD-pRNC[Thioether+DEA] was loaded with DID to form
cRGD-pRNC[Thioether+DEA]/DID, and Man-pRNC[Thioether+DEA] was
encapsulated in DIR to form Man-pRNC[Thioether+DEA]/DIR (Fig. [171]5a).
Fig. 5. Investigation of mixed nanoformulations for targeting both tumor
cells and TAMs in vivo.
[172]Fig. 5
[173]Open in a new tab
a Schematic illustration of mixed nanoformulation preparation, drug
administration and analysis. b In vivo fluorescence imaging after tail
vein injection of different formulations for cRGD targeting
investigation. c Quantitative analysis of fluorescence signals at 24 h
post-treatment (n = 3 mice per group). d Representative ex vivo images
of tumor tissues after treatments at 24 h. e Representative flow
cytometric images and quantification of DID^+ in PD-L1^+ tumor cells in
vivo (n = 3 mice per group). f In vivo fluorescence imaging after tail
vein injection of different formulations for Man targeting
investigation. g Quantitative analysis of fluorescence signals at 24 h
(n = 3 mice per group). h Representative ex vivo images of tumor
tissues. i Representative flow cytometric images and
semi-quantification analysis of DIR^+ in F4/80^+ TAMs cells in vivo
(n = 3 mice per group). Data are presented as mean ± SD. Statistical
significance was calculated through one-way ANOVA for multiple
comparisons using a Tukey post-hoc test.
For the cRGD nanoformulations, after intravenous (i.v.) injection the
in vivo fluorescence intensity within the 24 h first increased and then
decreased, and it reached a maximum at 8 h (Fig. [174]5b, c). The
fluorescence intensity of the nanoformulations was higher than that of
free DID at all time points after i.v. injection. At 8 h, in vivo
fluorescence intensity of cRGD-pRNC[Thioether+DEA]@DID and
cRGD-pRNC[Thioether+DEA]@DID mix Man-pRNC[Thioether+DEA]@DIR was 3.97-
and 3.88-fold higher than that of free DID, respectively, thus
indicating that the nanoformulation improved tumor accumulation. The in
vivo fluorescence intensity of the cRGD decoration group was higher
than that of the pRNC[Thioether+DEA]@DID group, highlighting the good
in vivo targeting ability of cRGD. According to ex vivo results, the
fluorescence intensity of the nanoformulation in tumor tissues was
11.63-fold higher than that of free DID (Fig. [175]5d). To further
analyze the ratios of cRGD nanoformulations in the tumor cells,
single-cell analysis was performed. According to Fig. [176]5e, higher
ratios of cRGD-pRNC[Thioether+DEA]@DID (13.27 ± 0.47%) and
cRGD-pRNC[Thioether+DEA]@DID mix Man-pRNC[Thioether+DEA]@DIR
(17.73 ± 0.85%) were detected and were 2.2- to 2.8-fold higher than
that of pRNC[Thioether+DEA]@DID (7.80 ± 0.84%) and free DID
(4.70 ± 0.50%) (Fig. [177]5e). To further verify if
cRGD-pRNC[Thioether+DEA] could target tumor cells, pRNC[Thioether+DEA]
and cRGD-pRNC[Thioether+DEA] were loaded with DID. According to the
immunofluorescence results presented in Supplementary Fig. [178]42,
cRGD-pRNC[Thioether+DEA]@DID exhibited a strong colocalization signal
(yellow color) compared to that of the pRNC[Thioether+DEA]@DID group
when tumor cells were stained with Alexa 488-PD-L1 (green). Red colors
represented DID and indicated the good tumor targetability of cRGD
modification. Negligible co-localization was observed in the free
DID-treated group.
The in vivo tumor accumulation and TAMs targetability of the
cRGD-pRNC[Thioether+DEA]@DID mix Man-pRNC[Thioether+DEA]@DIR were then
investigated. Compared to free DIR and pRNC[Thioether+DEA]@DIR, the in
vivo fluorescence intensity of Man-decorated nanoformulations was
effectively strengthened after i.v. injection, reaching a maximum at
8 h, and this was 3.88-fold higher than that of free DIR (Fig. [179]5f,
g). The ex vivo images also indicated similar results, where the
Man-pRNC[Thioether+DEA]@DIR and cRGD-pRNC[Thioether+DEA]@DID mix
Man-pRNC[Thioether+DEA]@DIR group were 3.67- and 4.20-fold higher than
the pRNC[Thioether+DEA]@DIR group, thus indicating the targetability of
Man (Fig. [180]5h). After single cell analysis of tumor tissue via FCM,
the ratios of Man-pRNC[Thioether+DEA]@DIR and
cRGD-pRNC[Thioether+DEA]@DID mix Man-pRNC[Thioether+DEA]@DIR in TAMs
were 12.90 ± 0.53% and 12.73 ± 0.51%, respectively, and this was
1.67-fold higher than that of pRNC[Thioether+DEA]@DIR (7.73 ± 0.86%),
DIR (3.32 ± 0.62%), and DID mix DIR (Fig. [181]5i). To further explore
the in vivo targetability of the Man nanoformulation in TAMs,
Man-pRNC[Thioether+DEA]@FITC was intravenously injected into LLC
tumor-bearing C57BL/6 mice, and this was followed by tumor tissue
extraction for immunofluorescence analysis. As presented in
Supplementary Fig. [182]43, Man-pRNC[Thioether+DEA]@FITC exhibited the
strongest co-localization (yellow color) when green colors represented
FITC and red colors represented TAMs, thus indicating that
Man-pRNC[Thioether+DEA] could efficiently target macrophages in vivo.
In vivo antitumor immune response activation
The cRGD- mix Man-pRNC[Thioether+DEA]/R848-mediated in vivo antitumor
immune response that recognizes high antitumor activity was then
investigated (Fig. [183]6a). As presented in Fig. [184]6b, c and
Supplementary Fig. [185]44, the expression of CD80 on the TAMs surfaces
increased, while that of CD206 decreased in mice after cRGD- mix
Man-pRNC[Thioether+DEA]@R848 (G7), Man-pRNC[Thioether+DEA]@R848 (G5)
treatment, or Man-pRNC[Thioether+DEA] (G2) alone, thus indicating that
R848 induced TAMs polarization and that the mixed formulations did not
affect the polarization ratio. Moreover, IL-12 levels increased in the
serum, while IL-10 levels decreased in mice in response to cRGD- mix
Man-pRNC[Thioether+DEA]@R848 (G7) (Fig. [186]6g, h, Supplementary
Fig. [187]45). As presented in Fig. [188]6d and Supplementary
Fig. [189]46a, the Foxp3^+ Tregs ratio notably decreased in mice
treated with cRGD- mix Man-pRNC[Thioether+DEA]@R848 (G7), thus
indicating its ability to remodel the TME.
Fig. 6. In vivo antitumor immune response of cRGD- mix
Man-pRNC[Thioether+DEA]@R848 with ICD inducement, TAMs polarization, Tregs
decrement and CD8^+/CD4^+ proliferation.
[190]Fig. 6
[191]Open in a new tab
a Schematic timeline of the experimental design to evaluate the in vivo
immune response activation. b, c Representative flow cytometric images
and quantitative analysis to show TAM polarization. d Representative
flow cytometric analysis gating on CD25^+ cells and quantification of
Foxp3^+ Tregs in tumors. e, f Representative flow cytometric analysis
and quantification of CD8^+ and CD4^+ T cells gating on CD3^+ cells. g,
h IL-10 and IL-12 levels of mouse serum after different treatments. i
Representative immunofluorescence images of tumors showing CD3^+CD4^+ T
cell infiltration. Scale bar = 50 μm. j Representative
immunofluorescence images of tumors showing CRT exposure. Scale bar =
50 μm. Experiments in (i) and (j) were repeated three times
independently with similar results. Data are presented as mean ± SEM
(n = 3 mice per group). Statistical significance was calculated
through one-way ANOVA for multiple comparisons using a Tukey post-hoc
test.
As presented in Fig. [192]6e, f and Supplementary Fig. [193]46b, c,
cRGD-pRNC[Thioether+DEA] (G3) and Man-pRNC[Thioether+DEA] (G2) elicited
an increase in the CD8^+ and CD4^+ T cell ratios compared to that of
PBS. Higher CD8^+ and CD4^+ T cell ratios were detected in both the
cRGD- mix Man-pRNC[Thioether+DEA]@R848 (G7) (CD8^+: 20.73 ± 3.82%,
CD4^+: 30.9 ± 4.37%) and cRGD- mix Man-pRNC[Thioether+DEA] (G4) (CD8^+:
14.7 ± 3.48%, CD4^+: 18.07 ± 3.10%) groups, thus indicating that immune
adjuvant of R848 could notably strengthen the in vivo immune response
(Supplementary Fig. [194]46, [195]47). CD8^+ and CD4^+ T cell
infiltration was observed in the mixed nanoformulation group based on
immunofluorescence staining results (Fig. [196]6i, Supplementary
Fig. [197]48). Additionally, TNF-α levels in serum was elevated in mice
with cRGD- mix Man-pRNC[Thioether+DEA]@R848 (G7) treatment, thus
confirming the systemic immune response activation (Supplementary
Fig. [198]49). To explore if cRGD-pRNC[Thioether+DEA] can induce tumor
ICD, tumor tissue sections from mice treated with different
nanoformulations were stained and characterized using CLSM. The results
demonstrated that cRGD-pRNC[Thioether+DEA] (G3) caused tumor ICD
following CRT exposure (Fig. [199]6j) and HMGB1 release (Supplementary
Fig. [200]50), thus indicating ICD induction.
To explore the possible clinical translation, the peripheral “vaccine”
ability was then investigated by separately loading OVA[257-264]
peptide and mRNA into cRGD- mix Man-pRNC[Thioether+DEA] to
self-assemble into nanovaccine for antigen-specific T cell and
durability response monitoring. As presented in Supplementary
Fig. [201]51, both cRGD- mix Man-pRNC[Thioether+DEA]@OVA and cRGD- mix
Man-pRNC[Thioether+DEA]@mRNA elicited antigen-specific T cell responses
with 1.44- and 1.67-fold higher CD8^+Tetramer^+ T cell ratios detected
compared to that of PBS on day 7 post-treatment. Even on day 14, high
ratios were observed, thus demonstrating durable responses and
suggesting that the intelligent nanoplatform possesses potent clinical
translation potential.
In vivo antitumor activity of cRGD- mix Man-pRNC[Thioether+DEA]@R848
The in vivo antitumor activity of cRGD- mix
Man-pRNC[Thioether+DEA]@R848 was investigated, and this was the final
purpose of this project. The detailed timeline is presented in
Fig. [202]7a. As presented in Fig. [203]7b and c, B16F10 tumor volume
growth was notably inhibited in mice with cRGD- mix
Man-pRNC[Thioether+DEA]@R848 (G7) treatment, while partial tumor volume
growth suppression was observed in the cRGD-pRNC[Thioether+DEA]@R848
(G5) group. Negligible changes in body weight were observed
(Fig. [204]7d). When the tumor volume increased to 2000 mm^3 on day-20
post-inoculation, the mice were euthanized, and the tumor tissue and
normal organs (e.g., heart, liver, spleen, lung, and kidney) were
extracted. As presented in Fig. [205]7e and f, the tumor volume and
weight in mice treated with cRGD- mix Man-pRNC[Thioether+DEA]@R848
prominently decreased to levels that were 7.5-fold lower than that of
PBS.
Fig. 7. In vivo antitumor activity of cRGD- mix Man-pRNC[Thioether+DEA]@R848
in B16F10 tumor model.
[206]Fig. 7
[207]Open in a new tab
a Schematic of treatment timeline in B16F10 tumor-bearing mice. b, c
Average and individual tumor growth curves after different treatments
(n = 5 mice per group). d Body weight changes within 20 days after
inoculation (n = 5 mice per group). e, f Images and weight of tumor
tissues collected on day 20 after different treatments (n = 5 mice per
group). g Representative flow cytometry images and quantification of
mature DCs in LNs (n = 3 mice per group). h Representative flow
cytometric analysis and quantification of CD8^+ and CD4^+ T cells
gating on CD3^+ cells (n = 3 mice per group). i Representative flow
cytometric analysis gating on CD47^+ cells in tumors (n = 3 mice per
group). j Representative flow cytometric analysis gating on PD-1^+CD8^+
cells in tumors (n = 3 mice per group). k Representative
immunofluorescence images of Foxp3^+ cells in tumors. Three times was
repeated independently with similar results. Scale bar = 50 μm. Data
are presented as mean ± SD. Statistical significance was calculated
through one-way ANOVA for multiple comparisons using a Tukey post-hoc
test.
To investigate if cRGD- mix Man-pRNC[Thioether+DEA]@R848 could activate
DCs, the ratio of mature DCs in the lymph nodes was detected by FCM. As
presented in Fig. [208]7g, the ratio of CD80^+CD86^+ DCs
(31.03 ± 0.47%) in mice with cRGD- mix Man-pRNC[Thioether+DEA]@R848
(G7) treatment significantly increased to levels that were 1.79- and
3.97-fold higher than those of cRGD- mix Man-pRNC[Thioether+DEA] (G4)
(14.00 ± 1.02%) and PBS (G1) (7.81 ± 0.84%). It was observed that the
highest CD8^+ (15.8 ± 2.55%) and CD4^+ (4.11 ± 1.84%) T cell ratio was
measured in the cRGD- mix Man-pRNC[Thioether+DEA]@R848 (G7) group
(Fig. [209]7h, Supplementary Fig. [210]52). Notably, Foxp3^+ expression
decreased in mice treated with the cRGD- mix
Man-pRNC[Thioether+DEA]@R848 (G7) with alleviated green fluorescence,
thus demonstrating its ability to modulate the TME (Fig. [211]7k).
Remarkably, the cRGD- mix Man-pRNC[Thioether+DEA]@R848 (G7)-treated
mice displayed a significant decrease in CD47 expression that may
potentiate macrophage phagocytosis of the tumor (Fig. [212]7i). Thus,
cRGD- mix Man-pRNC[Thioether+DEA]@R848 can activate the host immune
response for high antitumor activity via in situ cancer vaccination and
TAM polarization. However, upregulated PD-1 expression on the T cell
surface was detected after cRGD- mix Man-pRNC[Thioether+DEA]@R848
treatment at the therapeutic point, and this may impede antitumor
efficacy at the late stage, thus indicating the necessity for immune
checkpoint blockade (Fig. [213]7j, Supplementary Fig. [214]53). The
normal organ tissues in the mice treated with the nanoformulations were
similar to those treated with PBS, and this revealed the superior
biocompatibility of the nanocarriers (Supplementary Fig. [215]54).
In vivo antitumor activity of large tumors after combination anti-PD-1 and
anti-CD47 treatment
The detailed timeline is presented in Fig. [216]8a for investigation of
the antitumor activity against large tumors via the combination of
cRGD- mix Man-pRNC[Thioether+DEA]@R848 and antibodies. As presented in
Fig. [217]8b and c, tumor volume growth was significantly suppressed in
mice treated with cRGD- mix Man-pRNC[Thioether+DEA]@R848 in combination
with anti-PD-1 and anti-CD47 (G5), ultimately resulting in the longest
median survival. Partial tumor volume inhibition was observed with
combined anti-PD-1 (G4) or anti-CD47 (G3) alone, with a median survival
time of 30 days (Fig. [218]8e). Negligible body weight changes were
observed after the different treatments, thus indicating good
biocompatibility (Fig. [219]8d). On day 18, normal organs and tumor
tissues were obtained from one mouse per group (Supplementary
Fig. [220]55), and negligible damage to the normal organs was observed.
As indicated by the H&E, TUNEL, and Ki67 staining results, most tumor
cell death was observed after combination treatment with anti-CD47 plus
anti-PD-1 (Fig. [221]8f-h).
Fig. 8. In vivo antitumor activity of cRGD- mix Man-pRNC[Thioether+DEA]@R848
after combination with antibodies in mice with a large initial B16F10 tumor
volume.
[222]Fig. 8
[223]Open in a new tab
a Schematic illustration of the treatment timeline. b, c Average and
individual tumor volume growth curves after different treatments (n = 7
mice per group). d Body weight changes of mice after treatments (n = 7
mice per group). e Survival curves of tumor-bearing mice post different
treatments (n = 6 mice per group). f H&E Ki67 and TUNEL images of tumor
slices. Scale bar = 50 μm. g, h Quantitative analysis of fluorescence
intensity of Ki67 and TUNEL. Data are presented as mean ± SD (n = 3
independent experiments). Statistical significance was calculated
through one-way ANOVA for multiple comparisons using a Tukey post-hoc
test (b) or log-rank (Mantel-Cox) test (e).
Discussion
Utilizing the host adaptive immune system to attack tumors is an
effective method for cancer immunotherapy, and inducing tumor ICD to
elicit host antitumor immunity is one of the most widely used
approaches. However, it generally requires external inducers such as
photosensitizers and chemotherapeutics, most of which are encapsulated
or conjugated in carriers requiring site-specific delivery, thereby
increasing the complexity of nanoplatforms. To resolve these issues, an
immunoactive cRGD-conjugated polymer with a tertiary amine and
thioether that can self-assemble into a pH-responsive nanocarrier
(cRGD-pRNC[DEA+Thiother]) was synthesized to directly induce melanoma
B16F10 ICD via metabolic regulation and the mediation of ER stress with
TAAs and DAMPs release. After encapsulation with the TLR7/8 agonist
R848, cRGD-pRNC[DEA+Thiother] formed an in situ cancer vaccine in vivo
that led to DC maturation, antigen presentation, CD8^+/CD4^+ T-cell
proliferation, and tumor volume growth inhibition. In situ tumor
vaccination was performed after tumor ICD to ensure temporal
orchestration. Additionally, the cRGD-pRNC[DEA+Thiother]/R848 used in
this study possessed very simple components that contributed to
clinical translation with good biocompatibility, easy feasibility, high
tumor accumulation, and satisfactory therapeutic efficacy. In addition
to its combination with R848 for in situ tumor vaccination, the
nanoplatform can encapsulate ferroptosis or pyroptosis inducers for a
combination of different cell death modalities that mediate tumor
eradication. However, certain issues in nanosystems should not be
ignored, including the large-scale synthesis and thorough purification
of polymers.
Immunosuppressive factors such as TAMs are important components of
tumor tissues and are key roadblocks to cancer immunotherapy.
Therefore, it is necessary to remodel the TME. The same polymeric
skeleton as that of cRGD-pRNC[DEA+Thiother] was used here, but cRGD was
decorated with Man on the nanocarrier surface. The nanocarriers
specifically induced melanoma B16F10 ICD but not macrophages via dose
adjustment. After loading with R848, Man-pRNCs induced TAMs
polarization to the M1 phenotype with antitumor activity. In
particular, the composite nanoplatform cRGD- mix
Man-pRNC[DEA+Thiother]/R848 was able to separately target tumor cells
for in situ cancer vaccination and TAMs for polarization and
spatiotemporal orchestration of adaptive and innate immune response
activation. In addition to TAM polarization, TAM autophagy inhibition
and depletion can also be investigated in TME modulation, and this may
be another direction for future research.
In summary, we designed a pH-responsive polymeric nanoplatform in which
the nanocarrier could directly induce B16F10 ICD via inducing oxidative
phosphorylation, increasing mtROS production, and upregulating CHOP
that in turn induced ER stress. After decoration with cRGD and Man,
they can separately target tumor cells for in situ cancer vaccination
and TAMs for polarization via TME modulation. Superior antitumor
activity and immune response activation highlight the advantages of
utilizing the same polymeric skeleton for multiple functional
combinations. The prolonged median survival indicates the potential to
exert immune checkpoint blockade efficacy. This study provides insights
into utilizing immunofunctional polymers to form versatile nanocarriers
for potent cancer immunoefficacy that possess excellent clinical
translation potential due to their simple synthesis and design.
Methods
Ethical statement
Our research complies with all relevant ethical regulations. All animal
studies were performed under the approved protocol of Zhengzhou
University Animal Care and Use Committee (ZZU ACUC syxk (yu)
2018-0004).
Materials
Methoxy-poly(ethylene glycol)amine (MeO-PEG-NH[2], M[w] = 5.0 kg/mol)
and amino-poly (ethylene glycol)acid (NH[2]-PEG-COOH, Mw = 5.0 kg) were
purchased from Ponsure Biological (Shanghai, China). The compound
4-Cyano-4-(Phenylcarbonothioylthio) pentanoic acid N-succinimidyl ester
(CPPA, 98%) was purchased from Tokyo Chemical Industry Co., Ltd.
(Tokyo, Japan). Methyl methacrylate (MMA, 99.0%), propargylamine (98%),
3-mercaptopropionic acid (98%), 2-(diethylamino)ethyl methacrylate
(DEA, 99%), 2, 2’-azobis(2-methylpropionitrile) (AIBN, 99%),
N-hydroxysuccinimide (NHS, 98%),
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC·HCl,
98.5%), 4-dimethylaminopyridine (DMAP, 99%), and diethylaminoethanol
were purchased from Macklin (Shanghai, China). Methacryloyl chloride
(95% purity) was purchased from Aladdin (Shanghai, China). The compound
2-Mercaptoethanol (99%) was purchased from GEN-VIEW SCIENTIFIC INC, and
4-Aminophenyl-alpha-D-mannopyranoside (Mannose, 98%) and
cyclo(RGD-DPhe-K) (cRGDfK, 92%) were purchased from Top-peptide Co.,
Ltd. (Shanghai, China). Benzoin dimethyl ether (DMPA) was purchased
from Sigma-Aldrich. ER-Tracker Green, Golgi-Tracker Green, LysoTracker
Green, and MitoTracker Green were purchased from Beyotime. The ATP
Assay Kit (S0026) was purchased from Beyotime (Shanghai, China).
Thiazolyl blue tetrazolium bromide MTT (98%) was purchased from
Solarbio Life Sciences (Beijing, China). RMPI-1640, phosphate-buffered
saline (PBS), and 0.25% trypsin were purchased from Pricellella (Wuhan,
China). The mouse HMGB1 ELISA Kit was purchased from Enzyme-Linked
Biotechnology (Shanghai, China). The mouse TNF-α ELISA Kit was
purchased from Yuchun Biology (Shanghai, China). PE anti CD11c, PE anti
CD3, APC anti CD8a, APC anti CD86, Percp Cy5.5 anti CD4, Percp Cy5.5,
anti-CD80, FITC anti CD206, and Alexa Fluor^Ⓡ488 anti CRT were
purchased from BioLegend. Anti-elF2α (phosphor S52, 1:1000, ab227593,
Abcam), anti-PERK (phospho T982, 1:1000, ab192591, Abcam), and
anti-CHOP (1:1000) were purchased from Abcam. DNAse and collagenase
were purchased from Thermo Fisher Scientific. Cytokine IL-4, IFN-γ, and
recombinant mouse M-CSF was purchased from novoprotein (Shanghai,
China), and lipopolysaccharide (LPS) was purchased from Solarbio
(Beijing, China). IL-10, IL-12, and TNF-α ELISA Kits were purchased
from Lianke Biology (Hangzhou, China).
Characterization
The molecular weights of polymers were measured using a hydrogen
nuclear magnetic resonance (^1H NMR) instrument (Germany Brker). The
morphology of the nanoparticles was characterized using transmission
electron microscopy (TEM, Thermo Scientific/Talos L120CG2). The size
and zeta potential of the nanoparticles were measured using a Malvern
Nanosize Analyzer (ZEN-3600). Cytokine levels were tested via ELISA kit
and a microplate reader (Biotek Synergy H1, USA). Drug-loading content
(DLC) and drug-loading efficiency (DLE) were measured using a
fluorospectrophotometer. Cellular internalization, tumor ICD, and in
vivo T-cell proliferation and infiltration were characterized using
laser scanning confocal microscopy (CLSM, LEICA TCS SP8 STED) and flow
cytometry (Accuri C6 Plus). In vivo near-infrared (NIR) imaging was
performed using an animal multimodel imaging analyzer (IVIS Lumina XRMS
Series). The relative molecular weights of the polymers were determined
by gel permeation chromatography (GPC) using a Waters 1515 (Waters,
USA).
Synthesis and characterization of PEG-PMMA
The di-block copolymer PEG-PMMA was obtained through RAFT
polymerization using MeO-PEG-CPPA (also termed PEG-CPPA) and monomer
MMA. Prior to this, the macro-RAFT agent PEG-CPPA was synthesized by
the amidation reaction of PEG-NH[2] and NHS-CPPA as indicated in a
previous report^[224]48. Briefly, in the existence of the nitrogen
(N[2]) atmosphere, the solution of PEG-CPAA (100 mg, 0.02 mmol),
monomer MMA (200 mg, 2 mmol), and the initiator AIBN (0.49 mg,
0.003 mmol) in 1, 4-dioxane was reacted in a sealed flask after placing
it in an oil bath (70 ^oC) for 48 h. After precipitation,
centrifugation, and vacuum desiccation, the copolymer PEG-PMMA was
obtained at a 67% yield. According to the ^1H NMR results, the
molecular weight of the diblock copolymer is 5.0–10.0 kg/mol.
Synthesis of PEG-PMMA-PDEA
The triblock copolymer PEG-PMMA-PDEA was obtained through reversible
addition-fragmentation chain transfer (RAFT) polymerization using
PEG-PMMA and the monomer DEA. Briefly, under an N[2] atmosphere, the
solution of PEG-PMMA (64.5 mg, 0.0043 mmol), monomer DEA (19.96 mg,
0.108 mmol), and initiator AIBN (0.106 mg, 0.0006 mmol) in 1, 4-dioxane
was reacted in a sealed flask that was placed in an oil bath (70 ^oC)
for 48 h. After precipitation, centrifugation, and vacuum desiccation,
the copolymer PEG-PMMA-PDEA was obtained at a 78% yield. According to
the ^1H NMR results, the molecular weight of the triblock copolymer was
5.0-10.0-3.8 kg/mol.
Synthesis of N-propargyl methacrylamide (PPMA)
Small-molecule PPMA was obtained via amidation of methacryloyl chloride
and propargylamine. In detail, propargylamine (2.057 g, 37.4 mmol) and
DMAP (463.6 mg, 3.8 mmol) were fully dissolved in dichloromethane
(DCM). In an ice-water bath under an N[2] atmosphere, a solution of
DMAP and methacryloyl chloride was added dropwise to the propargylamine
reaction for 24 h at room temperature. The product was purified using a
silica gel column (mobile phase: n-hexane: ethyl acetate = 5/1).
According to ^1H NMR result, pure PPMA was obtained. The yield was 25%.
Synthesis of PEG-PMMA-P(PPMA-ME)
The triblock copolymer PEG-PMMA-PPPMA was synthesized via RAFT
polymerization to obtain the graft copolymer PEG-PMMA-P (PPMA-ME).
Briefly, PEG-PMMA (100 mg, 0.0067 mmol), PPMA (34 mg, 0.276 mmol), and
AIBN (0.164 mg, 0.001 mmol) were dissolved in 1, 4-dioxane and added to
a flask under an N[2] atmosphere. After further N[2] ventilation for
30 min, the flask was sealed and placed in an oil bath (70 °C) reaction
for 48 h. After ice diethyl ether precipitation, filtration, and vacuum
drying, PEG-PMMA-PPPMA was successfully acquired, and the molecular
weight was 5.0-10.0-6.3 kg/mol according to ^1H NMR result. The yield
was 52%.
PEG-PMMA-P(PPMA-ME) was obtained via a click reaction between
PEG-PMMA-PPPMA and mercaptoethanol. Briefly, PEG-PMMA-PPPMA (41.54 mg,
0.00195 mmol), mercaptoethanol (7.59 mg, 0.0973 mmol), and DMPA
(6.23 mg, 0.0243 mmol) were dissolved in N, N-dimethylformamide (DMF)
and added into a flask with UV laser irradiation (2 h, wavelength:
350 nm). After ice-cold diethyl ether precipitation, filtration, and
vacuum desiccation, the graft copolymer was successfully obtained at an
84% yield, and the graft ratio of mercaptoethanol was 100% according to
the ^1H NMR result. The molecular weight of PEG-PMMA-P(PPMA-ME) was
5.0-10.0-10.4 kg/mol.
Synthesis of PEG-PMMA-P(PPMA-Cy5)
PEG-PMMA-P(PPMA-Cy5) was obtained via a click reaction between
PEG-PMMA-PPPMA and Cy5-N[3]. Briefly, PEG-PMMA-PPPMA (20 mg,
0.001 mmol) and Cy5-N[3] (0.38 mg, 0.0006 mmol) were dissolved in DMF.
CuSO[4] (0.005 mg, 3.13 × 10^−5 mmol) was dissolved in deionized water
(1.68 μL) and then added into the DMF solution. After the reaction
mixture was incubated under an N[2] atmosphere for 30 min, sodium
ascorbate (NaAS) (0.026 mg, 0.0001 mmol) dissolved in deionized water
(21.7 μL) was added to the above reaction under N[2]. After reacting
for 24 h at room temperature, the product was subjected to DMF and
water dialysis and lyophilization. The yield was 74%.
Synthesis of PEG-PMMA-P(PPMA-MPA-DEA)
To obtain PEG-PMMA-P(PPMA-MPA-DEA), PEG-PMMA-P(PPMA-MPA) was
synthesized via a click reaction of PEG-PMMA-PPPMA and
mercaptopropionic acid. This was similar to the synthesis procedure for
PEG-PMMA-P(PPMA-ME) with the exception that mercaptoethanol was
replaced with mercaptopropionic acid. The molecular weight of
PEG-PMMA-P(PPMA-MPA) was 5.0-7.3-14.5 kg/mol according to the ^1H NMR
result. The yield was 85%.
PEG-PMMA-P(PPMA-MPA-DEA) was obtained via the esterification of
PEG-PMMA-P(PPMA-MPA) and diethylaminoethanol. In detail,
PEG-PMMA-P(PPMA-MPA) (112.5 mg, 0.0042 mmol), EDC·HCl (30.19 mg,
0.157 mmol), and DMAP (12.8 mg, 0.105 mmol) were separately dissolved
in DMF. Under an N[2] atmosphere, EDC·HCl and DMAP were added into
PEG-PMMA-P(PPMA-MPA) reaction for 30 min, and this was followed by DEA
addition. After reacting for 24 h at room temperature, the product was
subjected to DMF and water dialysis and lyophilization. The yield was
74%. According to ^1H NMR results, the molecular weight was
5.0-7.3-18.4 kg/mol.
Preparation and characterization of nanoparticles
All nanoparticles were prepared using the solvent-exchange method.
Briefly, polymers with different functional groups were dissolved in
THF at a concentration of 5 mg/mL, and 100 μL were then added into PBS
buffer (900 μL, 10 mM) for a uniform dispersion. After volatilization
and dialysis (MWCO = 3500) to remove the organic solvent, nanoparticles
were successfully prepared, and their sizes and zeta potentials were
measured by DLS. Their morphologies were characterized by TEM. Briefly,
the nanoparticles were added to a copper mesh with a carbon film and
left overnight. After removing the excess nanoparticles, images were
captured using TEM. It should be noted that nanoparticles
self-assembled from the polymer PEG-PMMA were termed as
nanocarrier[MMA](NC[MMA]), and self-assembly from PEG-PMMA-PDEA,
PEG-PMMA-PPPMA, PEG-PMMA-P(PPMA-hydroxyl), and PEG-PMMA-P(PPMA-DEA)
were named pH responsive nanoparticle[DEA] (pRNC[DEA]), NC[yne],
NC[Thioether], and pRNC[Thioether+DEA] For the investigation of pH
responsiveness, nanoparticles were placed in acetate buffer (10 mM,
pH 5.0) and PB buffer (pH 7.4, 10 mM), and their sizes were monitored
by DLS at 1, 12, and 24 h.
Cell culture
Macrophage RAW264.7 cells, Pan02 cells and U87-MG cells were separately
cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10%
fetal bovine serum (FBS) and 1% streptomycin-penicillin, and they were
cultured in an incubator (37 ^oC, 5% CO[2]). Melanoma B16F10 cells, 4T1
cells, MC38 cells, LLC cells and DC were separately cultured in
RPMI-1640 medium containing 10% FBS and 1% streptomycin-penicillin, and
they were cultured in an incubator (37 ^oC, 5% CO[2]). All cell lines
were obtained from American Type Culture Collection (ATCC). These cell
lines were authenticated by Wuhan Pricella Biotechnology (Wuhan, China)
using STR analysis. Specifically, 20 STR loci were amplified using
using Microreader 21 ID System. The PCR products were detected by
GenReader 7010, and the results were analyzed by GeneMapper Software6
and compared with ExPASy database. All cell lines were tested negative
by using Myco-Lumi Luminescent Mvcoplasma Detection Kit for mycoplasma
contamination.
Animals
C57BL/6 mice (female, 6 ~ 8 weeks, 18–20 g) were used to construct a
melanoma model. All animals were housed in individually ventilated
cages at Zhengzhou University, subjected to a 12-h light-dark cycle,
and maintained at 22 °C with humidity levels between 30% to 70%. Tumor
length and width were measured using a Vernier caliper every two days,
and the tumor volume curve was recorded. The maximal tumor volume
(2000 mm^3) was permitted by the institutional animal care and use
committee. As for results in Fig. [225]7c, at the second last time
point (day-18 post-inoculation), the tumor volume in all mice was less
than 2000 mm^3 and the tumor growth was continued to monitor. At the
day-20 post-inoculation, the tumor volume in one mouse of G1 reached
2000 mm^3 thereby being chosen as the endpoint. All the tumor-bearing
mice were euthanized at that time point for the immune response
analysis. The tumor volume (V) was calculated using the following
formula: V = ½ × Length × Width^[226]2.
Screening of the polymer selectively inducing B16F10 immunogenic cell death
Polymer-mediated ICD was explored using CRT exposure and HMGB1 and ATP
release assays. For CRT exposure, B16F10 cells (2.0 × 10^5/well) were
seeded into six-well plate and cultured overnight. NC[MMA], pRNC[DEA],
NC[yne], NC[Thioether], and pRNC[Thioether+DEA] (concentrations of all
nanoformulations were 50 μg/mL) were separately added into each well
incubation for 48 h. After washing with PBS three times and then
digestion and centrifugation, the cells were stained with Alexa
Fluor^Ⓡ488 anti CRT (400×, 40 min), and this was followed by PBS
washing and centrifugation. Finally, the cells were suspended in PBS
(0.5 mL each sample), stained with propidium iodide (PI), incubated at
room temperature for 10 min, and detected by flow cytometry, processed
using FlowJo software (version 10.9.0).
To characterize HMGB1 release, cells were seeded into 24-well plates
and cultured overnight, and this was followed by treatment with
NC[MMA], pRNC[DEA], NC[yne], NC[Thioether], or pRNC[Thioether+DEA].
After nanoformulation treatment for 48 h, PBS washing (× 3), digestion,
and centrifugation, the cells were stained with Alexa Fluor^Ⓡ488 anti
HMGB1 (200×, 40 min), and this was followed by PBS washing. Then,
mounting media containing DAPI was added to the cells. The specimens
were covered with coverslips and sealed with nail polish. Cell images
were captured using a confocal laser-scanning microscope (CLSM),
processed using LAS X 4.4 software. After the different treatments, the
supernatants were collected to detect HMGB1 release via ELISA
characterization.
ATP secretion by B16F10 cells after the different treatments was tested
using an ATP Assay Kit (S0026). B16F10 cells (2.0 × 10^5/well) were
seeded into six-well plates and cultured overnight. NC[MMA], pRNC[DEA],
NC[yne], NC[Thioether], and pRNC[Thioether+DEA] were separately added
into each well for 24 h. A total of 30 μL of supernatant from the cell
culture medium was added to 70 μL of ATP assay working solution, and
the RLU value was measured by enzyme-linked immunosorbent assay within
30 min. The total protein concentration in the six-well plates was
determined using a BCA kit. The amount of ATP released per unit protein
concentration was compared among the different groups.
Concentration and time dependence of pRNC[Thioether+DEA]-mediated B16F10 ICD
For CRT exposure at the same time points with different concentrations,
B16F10 cells (2.0 × 10^5/well) were seeded into six-well plates and
cultivated overnight. pRNC[Thioether+DEA] (the concentrations of the
nanoformulations were 0, 25, 50, 100, 200, and 300 μg/mL) were
separately added into each well for 48 h incubation. After washing with
PBS (× 3), digestion, and centrifugation, the cells were stained with
Alexa Fluor^Ⓡ488 anti CRT (400×, 40 min), and this was followed by PBS
washing and centrifugation. The cells were then suspended in PBS
(0.5 mL) and stained with PI. After 10 min, the cells were washed with
PBS, centrifuged, suspended in PBS, and analyzed by flow cytometry.
For CRT exposure at the same concentration and at different time
points, B16F10 cells (2.0 × 10^5/well) were seeded into six-well plates
and cultivated overnight. pRNC[Thioether+DEA] (100 μg/mL) was
separately added into each well for different incubation time. After
washing with PBS (× 3), digestion, and centrifugation, the cells were
stained with Alexa Fluor^Ⓡ488 anti CRT (400×, 40 min), and this was
followed by PBS washing and centrifugation. The cells were then stained
with propidium iodide (PI), washed with PBS, suspended in PBS, and
analyzed by flow cytometry.
Organelle targetability investigation
B16F10 cells (2.0 × 10^4) were seeded into a dish and cultured for
24 h. Cy5-labeled pRNC[Thioether+DEA] (Cy5-pRNC[Thioether+DEA]) was
added and incubated for 2 and 4 h, respectively. After washing (PBS ×
3), cells were separately stained with ER-Tracker Green, Golgi-Tracker
Green, Lyso-Tracker Green, and Mito-Tracker Green (30 min). After PBS
washing (× 3) again, cells were stained with Hoechst 33342 (5 μg/mL,
15 min). After further PBS washing, fresh media (200 μL) was added to
each dish, and images were captured by CLSM.
Mechanisms investigation of the polymer-mediated ICD
The underlying mechanisms were explored by sequence analysis, flow
cytometry, CLSM and WB characterization, and western blotting. For
RNA-sequence analysis, B16F10 cells (2.0 × 10^5/well) were seeded into
six-well plates and cultured overnight. PBS and pRNC[Thioether+DEA]
were added and incubated for 48 h. After removal of the culture media,
TRIzol (1 mL) was added to each well for cell digestion, and the cells
were transferred into RNase-free tubes. After 1–2 seconds in liquid
nitrogen, the samples were placed at −80 ^oC for 24 h. Interfering DNA
was removed, and RNA samples were prepared. An Illumina TruseqTM RNA
sample prep kit was used to construct the library that was then
sequenced. Bioinformatics analysis was performed on the sequencing
results.
Flow cytometry and CLSM were performed to determine intracellular ROS
and mtROS generation. For flow cytometry analysis, B16F10 cells (2.0 ×
10^5/well) were seeded into six-well plates and cultured overnight.
Cells were incubated with PBS, NC[MMA], and pRNC[Thioether+DEA] for
48 h. After PBS washing, DCFH-DA probe (1 mL, 5 μM in serum-free
RPMI-1640 medium) was added to each well and incubated for 30 min away
from light. After washing (PBS × 3), digestion, and centrifugation, the
cells were suspended in PBS (0.5 mL each sample) and detected by flow
cytometry. The generation of pRNC[Thioether+DEA]-mediated mtROS within
the tumor cells was observed via CLSM. B16F10 cells (2.0 × 10^4/well)
were seeded into a culture dish for 24 h. PBS, NC[MMA], and
pRNC[Thioether+DEA] were added and incubated for 48 h. After PBS
washing (× 3), cells were separately stained with BBcellProbe®OM08
(30 min) and Hoechst 33342 (5 μg/mL, 15 min). After further PBS
washing, fresh media (200 μL) was added to each dish, and images were
captured by CLSM.
For WB characterization, B16F10 cells (2.0 × 10^5/well) were seeded
into six-well plates and cultured overnight. PBS, NC[MMA], and
pRNC[Thioether+DEA] were added to each well and incubated for 48 h.
After washing with PBS, the cells were lysed using
radioimmunoprecipitation assay (RIPA) buffer, and the supernatant was
collected after centrifugation (10,000 g, 30 min). The protein content
was measured using a bicinchoninic acid kit. Proteins were separated by
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF)
membrane. For necrosis-related proteins, MLKL (1:1000, cat# 183770,
Abcam) was detected. For ER stress-related proteins, p-PERK (1:1000,
Cell Signaling Technology, cat# 3179S), p-elf2α (1:1000, Cell Signaling
Technology, cat# 9721S), CHOP (1:1000, cat.no.ab11419, Abcam), and
ATF-4 (1:1000, sc-390063, Santa Cruz Biotech) were used. For
pyroptosis-related proteins, cleaved caspase-1 (1:1000, Ala317,
Affinity) and N-GSDMD (1:1000, cat# 939701, BioLegend) were used.
Primary antibody solutions were prepared in a primary antibody dilution
buffer (EpiZyme). The reaction was performed overnight at 4 °C.
HRP-conjugated anti-rabbit and anti-mouse that were used as secondary
antibodies were added to the corresponding PVDF membranes at room
temperature for 1 h. The blot signals were visualized using an enhanced
chemiluminescent (ECL) reagent (Epizyme, China).
LDH detection
LDH secretion from B16F10 cells after different treatments was tested
using an LDH Microplate test kit (A020-2). B16F10 cells
(2.0 × 10^5/well) were seeded into six-well plates and cultured
overnight. PBS, NC[MMA], and pRNC[Thioether+DEA] were separately added
to each well and incubated for 24 h. Supernatants were collected and
measured according to the manufacturer’s instructions.
B16F10 cells death mediated by pRNC[Thioether+DEA]
Tumor cells death was determined by flow cytometry. First, B16F10 cells
(2.0 × 10^5/well) were seeded into a six-well plate and grown
overnight. PBS, NC[MMA], pRNC[Thioether+DEA],
pRNC[Thioether+DEA] + Z-VAD-FMK, pRNC[Thioether+DEA] +Nec-1s, and
pRNC[Thioether+DEA] + Fer-1 were separately added into each well for
48 h incubation. Cells were digested by trypsin and subsequently washed
with PBS (×3). Cell death was assessed by flow cytometry using an
Annexin V/PI cell assay kit (Biosharp) via the protocol provided by the
manufacturer.
Intracellular lipid peroxide detection
First, B16F10 cells (2.0 × 10^5/well) were seeded into a six-well plate
and cultured overnight. Phosphate-buffered saline (PBS), NC[MMA], and
pRNC[Thioether+DEA] were added to each well and incubated for 24 h.
After incubation for 24 h, the culture medium was removed, cells were
washed 3 times with PBS, and C11-BIODPY (1 mL, 10 μM, serum-free 1640
medium was used) was added to each well for 30 min incubation away from
light in a cell incubator. The cells were then digested with trypsin
and washed three times with PBS. The cells were suspended in PBS
(0.5 mL) and detected using flow cytometry.
Synthesis of Man (cRGD)-PEG-PMMA-PPPMA
Man-PEG-PMMA-PPPMA was obtained by the amidation of COOH-PEG-PMMA-PPPMA
and Man-NH[2]. The synthesis of COOH-PEG-PMMA-PPPMA was similar to that
of PEG-PMMA-PPPMA, and the molecular weights was determined by ^1H NMR
spectroscopy. To obtain Man-PEG-PMMA-PPPMA, NHS (0.489 mg, 0.0043 mmol)
and EDC·HCl (0.815 mg, 0.0043 mmol) were first added into the mixture
solution of COOH-PEG-PMMA-PPPMA (35 mg, 0.0028 mmol) and triethylamine
(TEA) with stirring for 1.5 h under N[2] to acquire NHS-PEG-PMMA-PPPMA.
In the ice water bath and N[2] atmosphere, NHS-PEG-PMMA-PPPMA was added
dropwise to a Man-NH[2] solution containing TEA. The reaction was then
placed in a water bath (30 ^oC) for 24 h under dark conditions. The
product was successfully obtained after dialysis and lyophilization
with a yield of 48%. The synthesis of cRGD-PEG-PMMA-PPPMA was similar
to that of Man-PEG-PMMA-PPPMA with the replacement of Man-NH[2] by
cRGD-NH[2], and the yield was 85%. cRGD (Man)-pRNC[Thioether+DEA] was
also prepared using a solvent-exchange method similar to that of
pRNC[Thioether+DEA] with a pre-mixture of the polymers
cRGD-PEG-PMMA-PPPMA (20 μL, 5 mg/mL) and PEG-PMMA-P(PPMA-Thioether-DEA)
(80 μL, 5 mg/mL). The size and morphology of cRGD-pRNC[Thioether+DEA]
was characterized by DLS and TEM, respectively.
Cytotoxicity investigation of Man-pRNC[Thioether+DEA] in RAW264.7 cells and
of cRGD-pRNC[Thioether+DEA] in B16F10 cells
Cytotoxicity was investigated by MTT assays. Briefly, RAW264.7 cells
and B16F10 cells (5.0 × 10^3/well) were seeded into 96-well culture
plates for 24 h. Man-pRNC[Thioether+DEA] or cRGD-pRNC[Thioether+DEA]
with different concentrations from low to high (0, 12.5, 25, 50, 100,
and 200 μg/mL) was separately added. After incubation for 48 h, MTT
solution (20 μL, 5 mg/mL) was added for 4 h incubation. DMSO (100 μL)
was added after supernatant aspiration. Absorbance at 570 nm was
measured using a microplate reader.
In vitro drug loading and release
Preparation of drug-loaded nanoparticles was also performed utilizing
the solvent-exchange method using only a pre-mixture of the polymer and
drug. In detail, the mixture of polymer PEG-PMMA-P(PPMA-DEA) (80 μL,
5 mg/mL), cRGD (Man)-PEG-PMMA-PPPMA (20 μL, 5 mg/mL) in THF (50 μL,
5 mg/mL), and R848 in DMF (10 μL, 5 mg/mL) was added dropwise into PBS
(900 μL) for a homogeneous dispersion. After volatilization and
dialysis to remove the organic solvent and unloaded drug, the
nanomedicine cRGD (Man)-pRNC[Thioether+DEA]@R848 was successfully
prepared, and its size was characterized by DLS. DLC and DLE were
measured using a fluorescence spectrophotometer and calculated
according to the following formulas:
[MATH: DLC=massofactualdrugencapsulation/massof(actualdrugencapsulation+polymer)*
100% :MATH]
1
[MATH: DLE=massofactualdrugencapsulation/massoftheoreticaldrugencapsulation*100%
:MATH]
2
In vitro drug release was investigated under specific conditions.
Briefly, cRGD (Man)-pRNC[Thioether+DEA]/R848 (each 0.5 mL) in PBS (pH
7.4, 10 mM, 150 mM NaCl) or acetate buffer (pH 5.0, 10 mM, 150 mM NaCl)
in a dialysis bag (MWCO = 12000) was placed in 25 mL of media and were
then placed in a shaking bed (37 ^oC, 200 rpm) (n = 3 independent
experiments). At different time points (1, 2, 4, 6, 9, 12, and 24 h),
5 mL of the medium was removed and replaced with the same volume of
fresh medium. Accumulation of R848 was detected using a fluorescence
spectrophotometer.
Targetability of cRGD-pRNC[Thioether+DEA] in B16F10 cells
The targetability of cRGD-pRNC[Thioether+DEA] was investigated using
flow cytometry and CLSM. For flow cytometry, B16F10 cells
(5.0 × 10^5/well) were seeded into a six-well plate and cultured
overnight, and this was followed by the addition of free FITC,
cRGD-pRNC[Thioether+DEA]@FITC, and pRNC[Thioether+DEA]@FITC. After
incubation for 10 or 30 min or for 1 h, the cells were subjected to
trypsin digestion, centrifugation, suspension in PBS, and detection by
flow cytometry.
For CLSM characterization, B16F10 cells were seeded into 24-well plates
containing cell slide cultures for 24 h. Free FITC,
cRGD-pRNC[Thioether+DEA]@FITC, and pRNC[Thioether+DEA]@FITC were added
separately and incubated for 10 min, 30 min, and 1 h. After PBS washing
(×3), cells were fixed for 15 min. After another PBS washing, cells
were stained with DAPI (5 μg/mL, 10 min) and then sealed using
coverslips onto microslides. After sealing with nail polish, images
were captured using a CLSM.
Targetability of Man-pRNC[Thioether+DEA] in RAW264.7 cells
Man-pRNC[Thioether+DEA] was also fabricated using a solvent-exchange
method similar to that of cRGD-pRNC[Thioether+DEA] and was
characterized by DLS and TEM. Its targetability was investigated by
flow cytometry and CLSM in a manner similar to that of
cRGD-pRNC[Thioether+DEA]. B16F10 cells were replaced with RAW264.7
cells.
In vitro DC maturation
DC maturation was explored by flow cytometry to characterize the marker
changes. Briefly, B16F10 cells (2.0 × 10^5/well) were seeded into
6-well plates and cultured overnight. PBS, NC[MMA],
cRGD-pRNC[Thioether+DEA], cRGD-pRNC[Thioether+DEA]@R848, and cRGD- mix
Man-pRNC[Thioether+DEA]@R848 were separately added into B16F10 cells
incubation for 48 h. The supernatant were then added into DC cells
(5.0 × 10^5/well) for another 24 h. After washing with PBS, trypsin
digestion, and centrifugation, the cells were stained with anti CD80,
anti CD86 (40 min), suspended in PBS, and analyzed by flow cytometry.
Cells treated with LPS (10 ng/mL) were used as the positive control.
In vitro macrophage polarization
Macrophage polarization was explored primarily by flow cytometry to
characterize marker changes on the cell surface and by ELISA to test
cytokine levels. For flow cytometry characterization, RAW 264.7 cells
(1.0 × 10^5/well) were seeded into 12-well plates and cultured
overnight. IL-4 (10 ng/mL) and Recombinant Mouse Macrophage
Colony-Stimulating Factor 1 (MCS-F1; 10 ng/mL) were added separately to
polarize M0 cells into the M2 phenotype. PBS, Man-pRNC[Thioether+DEA],
R848, Man-pRNC[Thioether+DEA]@R848, and cRGD- mix
Man-pRNC[Thioether+DEA]@R848 were separately added into M2 phenotype
cells (R848:1 μg/mL) for 4 h incubation. The original medium was
aspirated with fresh medium and incubated for another 20 h. After
washing with PBS, trypsin digestion, and centrifugation, the cells were
stained with anti CD80, anti CD206, and anti F4/80 (40 min), suspended
in PBS, and analyzed by flow cytometry. Cells treated with LPS
(10 ng/mL) and IFN-γ (10 ng/mL) were used to polarize M0 into the M1
phenotype as the positive control and CD80 single staining group.
Intracellular iNOS levels in RAW264.7 cells were detected by RT-qPCR.
Briefly, total RNA was isolated from RAW264.7 cells using the RNA Easy
Mini Plus Kit after R848, Man-pRNC[Thioether+DEA], and
Man-pRNC[Thioether+DEA]@R848 treatments. Two-step RT-qPCR was performed
on a Mastercycler EP Realplex (Eppendorf) using iScript and SsoAdvanced
SYBR Green Mix (Bio-Rad). The data were normalized to the reference
gene β-actin and compared to the control samples (n = 3 independent
experiments). Morphological hierarchical analysis was used to analyze
the data. Primers were purchased from Sangon Biotech with the sequence
are GTTCTCAGCCCAACAATACAAGA(5’ to 3’) and GTGGACGGGTCGATGTCAC(5’ to
3’).
In vivo NIR imaging and targeted internalization by tumor tissue and TAMs
For in vivo NIR imaging and targetability in tumor tissues, C57BL/6
mice were inoculated with LLC cells (1 × 10^6/mouse) in the right flank
to construct a tumor model. When the tumor volume reached approximately
150 mm^3, the mice were randomly divided into five groups that included
free DID (G1), free DID mix DIR (G2), pRNC[Thioether+DEA]@DID (G3),
cRGD-pRNC[Thioether+DEA]@DID (G4), and cRGD-pRNC[Thioether+DEA]@DID mix
Man-pRNC[Thioether+DEA]@DIR (G5) (n = 3 mice per group). When different
formulations were administered via intravenous (i.v.) injection, the
fluorescence intensity in tumor tissues were monitored via IVIS imaging
system at pre-set time points (2, 4, 8, 12, and 24 h). At 24 h, the
mice were euthanized, their organs were harvested (e.g., heart, liver,
spleen, lung, and kidneys), and tumors were extracted for ex vivo
imaging. Subsequently, tumor tissues were digested by collagenase IV
(50 U/mL) in 1640 medium containing with 2% FBS for 2 h (37 ^oC). The
supernatant was filtrated through a 70 μm filter membrane, and this was
followed by centrifugation (137 × g for 5 min) to acquire cell pellets.
The cells were then stained with FITC anti PD-L1 (400×, 40 min,
Elabscience), washed with PBS, centrifuged, resuspended in PBS, and
detected by flow cytometry.
TAMs targetability was performed in a manner similar to that of the
above tumor tissue targetability with formulations replaced by free DIR
(G1’), free DIR mix DID (G2), pRNC[Thioether+DEA]@ DIR (G3’),
Man-pRNC[Thioether+DEA]@DID (G4’), and cRGD-pRNC[Thioether+DEA]@DID mix
Man-pRNC[Thioether+DEA]@DIR (G5) and the antibody replaced by PE anti
F4/80.
In vivo ICD and TAM polarization
C57BL/6 mice were inoculated with B16F10 cells (1 × 10^6/mouse) in the
right flank to establish a melanoma tumor model. When the tumor volume
increased to approximately 100 mm^3 on day-6, the mice were randomly
divided into seven groups that included PBS (G1),
Man-pRNC[Thioether+DEA] (G2), cRGD-pRNC[Thioether+DEA] (G3), cRGD mix
Man-pRNC[Thioether+DEA] (G4), Man-pRNC[Thioether+DEA]@R848 (G5),
cRGD-pRNC[Thioether+DEA]@R848 (G6), and cRGD-mix
Man-pRNC[Thioether+DEA]@R848 (G7) (n = 3 mice per group)
(cRGD-pRNP[Thioether+DEA]: 1 mg/kg; Man-pRNP[Thioether+DEA]: 0.5 mg/kg;
R848: 0.1 mg/kg). At day-11 post-inoculation, serum was collected for
IL-10, IL-12, and TNF-α measurement by ELISA, and tumor tissue was
extracted for ICD and T cell ratio analysis. Partial tumor tissues were
subjected to mechanical obstruction, collagenase digestion, filtration,
staining with CD8/CD4/CD3, CD80, CD86, CD206, and Treg antibodies,
suspension in PBS, and flow cytometry. Partial tumor tissues were
embedded, sectioned, and subjected to immunofluorescence to
characterize CRT exposure, HMGB1 secretion, and CD8^+/CD4^+/CD3^+ T
cell infiltration using CLSM.
In vivo vaccine function of pRNC[Thioether+DEA]
B16F10 cells (1 × 10^6/ mouse) were inoculated subcutaneously into the
right side of C57BL/6 mice to establish a melanoma mouse model. When
the tumor volume reached approximately 100;mm^3 by day 6, the mice were
randomly divided into three groups that included PBS (G1),
pRNC[Thioether+DEA]@OVA (G2), and pRNC[Thioether+DEA]@mRNA (G3) (n = 3
mice per group) (pRNC[Thioether+DEA]: 5 mg/kg; OVA: 1 mg/kg; mRNA:
0.5 mg/kg). On days 7 and 14 after administration, peripheral blood
mononuclear cells were extracted from mice for tetrameric analysis. T
cells were extracted using a peripheral lymphocyte separation kit,
stained with a CD3/CD8/Tetramer antibody, resuspended in PBS, and
detected by flow cytometry.
In vivo antitumor activity of cRGD- mix Man-pRNP[Thioether+DEA]@R848
C57BL/6 mice were injected with B16F10 cells (1 × 10^6/mouse) in the
right flank to construct a melanoma tumor model. When the tumor grew to
~50 mm^3, the mice were randomly divided into seven groups that
included PBS (G1), Man-pRNC[Thioether+DEA] (G2),
cRGD-pRNC[Thioether+DEA] (G3), cRGD- mix Man-pRNP[Thioether+DEA] (G4),
Man-pRNC[Thioether+DEA]@R848 (G5), cRGD-pRNC[Thioether+DEA]@R848 (G6),
and cRGD-mix Man-pRNC[Thioether+DEA]@R848 (G7)
(cRGD-pRNP[Thioether+DEA]: 1 mg/kg; Man-pRNP[Thioether+DEA]: 0.5 mg/kg;
R848: 0.1 mg/kg) (n = 5 mice per group). Different formulations were
intravenously injected into tumor-bearing mice at 6, 9, and 12 days
post-inoculation. Tumor length and width were measured using a Vernier
caliper every two days, and the tumor volume curve was recorded. Body
weight was recorded every two days. The therapeutic end point was when
the tumor volume reached 2000 mm^3 with normal organ (e.g., the heart,
liver, spleen, lung, and kidneys) and tumor tissue extraction. Partial
tumor tissues were subjected to mechanical obstruction, collagenase
digestion, filtration, staining with CD8/CD4/CD3, CD80, CD86, CD206,
CD47, and PD-1 antibodies, suspension in PBS, and flow cytometry.
Partial tumor tissues were subjected to embedding, ice sectioning,
immunofluorescence staining, and Foxp3^+ Treg cell infiltration
characterization by CLSM.
In vivo antitumor activity of cRGD mix Man-pRNP[Thioether+DEA]@R848 in
combination with anti-PD-1 and anti-CD47
C57BL/6 mice were inoculated with B16F10 cells (1.5 × 10^6/mouse) via
subcutaneous injection to construct a melanoma tumor model. When the
tumor volume reached ~120 mm^3, mice were randomly divided into five
groups that included PBS (G1), cRGD mix Man-pRNC[Thioether+DEA]@R848
(G2), G2 plus anti-CD47 (G3), G2 plus anti-PD-1 (G4), and G2 plus anti
CD47 plus anti-PD-1 (G5) (cRGD-pRNP[Thioether+DEA]: 1 mg/kg;
Man-pRNP[Thioether+DEA]: 0.5 mg/kg; R848: 0.1 mg/kg; anti CD47:
2 mg/kg; anti PD-1: 2 mg/kg) (n = 7 mice per group). The
nanoformulations were administered on days 8, 11, and 14 via i.v. tail
injection three times when antibodies were intravenously injected on
days 9, 12, and 15. Tumor volume and mouse body weight were measured
every two days. On day 18 when the tumor volume increased to 2000 mm^3,
one mouse from each group was euthanized with harvesting of normal
organs, and the tumor tissue was extracted for H&E staining. Tumor
tissues were also used for TUNEL and Ki67 staining to investigate
apoptosis.
Statistical analysis
All quantitative data were collected from experiments performed in at
least triplicate and were expressed as mean ± SD/SEM. All statistical
analyses were performed using GraphPad Prism 10.0.0. Statistical
significance was calculated through one-way ANOVA for multiple
comparisons using a Tukey post-hoc test, two-tailed student’s t test
and the log-rank test. P-values and replicate n values refer to one-way
ANOVA and independent experiments, respectively, unless otherwise
stated. No statistical methods were used to determine the sample size.
Differences were considered significant at ^*P < 0.05, ^**P < 0.01,
^***P < 0.001, and ^****P < 0.0001 and not significant (ns) at
P ≥ 0.05.
Reporting summary
Further information on research design is available in the [227]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[228]Supplementary information^ (8.9MB, pdf)
[229]Peer Review File^ (6.7MB, pdf)
[230]Reporting Summary^ (5.1MB, pdf)
Source data
[231]Source Data^ (19.7MB, xlsx)
Acknowledgements